96-well Serum/Plasma Glycerol Kit Free Glycerol ... - Zen-Bio Inc.

96-well Serum/Plasma Glycerol Kit
Free Glycerol Detection
Cat# SGA-1
INSTRUCTION MANUAL
ZBM0043.00
STORAGE CONDITIONS
Reagents & Buffers: 4°C
Glycerol Standard: -20°C
Blank assay plates (96-well): Room Temperature
For in vitro Use Only
LIMITED PRODUCT WARRANTY
This warranty limits our liability to replacement of this product. No other warranties of any kind, expressed or implied, including
without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Zen-Bio, Inc. Zen-Bio, Inc.
shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the
inability to use this product.
ORDERING INFORMATION AND TECHNICAL SERVICES
Zen-Bio, Inc.
3200 Chapel Hill-Nelson Blvd., Suite 104
PO Box 13888
Research Triangle Park, NC 27709
Telephone (919) 547-0692
Facsimile (FAX) (919) 547-0693
Toll Free 1-866-ADIPOSE (866)-234-7673
Electronic mail (e-mail)
World Wide Web
information@zen-bio.com
http://www.zen-bio.com
Rev 8/15/2008 Page 1 of 6

INTRODUCTION
This kit is designed to accurately determine the amount of glycerol present in blood serum or plasma of
humans, mice, rats, and other animals in a 96-well format for increased throughput analysis. Blood can be
collected in plain evacuated tubes or in the presence of common anti-coagulants: sodium citrate, ammonium
oxalate and EDTA. NOTE: Heparin or Heparinized tubes should not be used because this will generate
inaccurate readings. Serum should be separated from clotted blood by centrifugation as soon as possible and
may be stored frozen (-20°C) prior to analysis.
PRINCIPLE OF THE ASSAY
Detection of Free Glycerol
Glycerol present in sample is phosphorylated by adenosine triphosphate (ATP) forming glycerol-1-
phosphate (G-1-P) and adenosine-5’-diphosphate (ADP) in the reaction catalyzed by glycerol kinase. G-1-P is
then oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate (DAP) and hydrogen peroxide
(H 2 O 2 ). A quinoneimine dye is produced by the peroxidase catalyzed coupling of 4-aminoantipyrine (4-AAP) and
sodium N-ethytl-N-(3-sulfopropyl)m-anisidine (ESPA) with H 2 O 2 , which shows an absorbance maximum at
540nm. The increase in absorbance at 540nm is directly proportional to glycerol concentration of the sample.
GLYCEROL + ATP
G-1-P + ADP
G-1-P + O 2 DAP + H 2 O 2
H 2 O 2 +4-AAP + ESPA
Quinoneimine dye + H 2 O
ITEMS INCLUDED IN THE KIT
ITEM DESCRIPTION Cap UNIT QTY STORAGE
Color
Assay Plate, Plate A 96-well assay plate, blank --- PLATE 2 -----
Dilution Buffer 12 ml --- BOTTLE 1 4°C
Glycerol Reagent A Reconstitute with 11.0 ml deionized water prior to use. --- BOTTLE 1 4°C
Tray For multi-channel pipetters, clear polyvinyl CLEAR EACH 2 -----
Glycerol standard Glycerol @ 1mM [Dilute with 200 l Dilution Buffer to ORANGE 50 l / 1 -20°C
make the 200 M glycerol standard; see page 5 for
recommended dilution scheme]
Other equipment/reagents required but not provided with the kit:
Multi-channel Pipet , single channel pipet and pipet tips
Plate reader with a filter of 540 nm
Incubator at 37 o C
Large gauge needle
Tubes for diluting standards
VIAL
Rev 8/15/2008 Page 2 of 6

ASSAY PROCEDURE
1. Prepare the glycerol standards as follows:
Briefly spin down the contents of the glycerol standard tube before reconstitution. Pipette 200 l of
Dilution Buffer into the 1 mM glycerol standard tube provided and mix well by vortexing. This
produces a diluted stock glycerol standard of 200 M. Pipette 125 l of dilution buffer into 6 tubes
(not provided). Using the newly diluted stock glycerol solution, prepare a dilution series as
depicted below. Mix each new dilution thoroughly before proceeding to the next. The 200 M
stock dilution serves as the highest standard, and the dilution buffer serves as the zero standard.
200 l 125 l 125 l 125 l 125 l 125 l 125 l
Dilution
Buffer
Std
200
M
100
M
50
M
25
M
12.5
M
6.25
M
3.125
M
2. Also at this time prepare the Glycerol Reagent A by adding 11.0 ml room temperature deionized water per
bottle and gently invert. DO NOT VORTEX! Use a pipet to ensure that the powder is completely
dissolved. Store at room temperature. If using a Reagent A solution previously prepared and stored at 2-
8 C, also bring to room temperature. Make sure there is enough Reagent A from one solution to treat all
the points in the assay. It may be necessary to combine solutions. Store in a light protected bottle.
Reconstituted Glycerol Reagent A is stable for 60 days refrigerated (2-8 C); store any remaining solution
refrigerated (2-8 C).
3. Add 20 l (or 10 - 25 l) of serum or plasma to a well of a blank plate for assessment of free glycerol. Add
30 l of dilution buffer to each well to total 50 l including serum or plasma sample. THIS RESULTS IN A
2.5x DILUTION OF YOUR SAMPLE (20 l in 50 l). Add 50 l of each standard to empty wells (use
another plate, if necessary).
4. Add the reconstituted Glycerol Reagent A solution to one of the disposable trays provided in the kit. Add
50 l of Reagent A to each well of the glycerol plate. Gently, pipet up and down once to mix. Pop the
bubbles using a large gauge needle or a clean pipet tip. The plate is then incubated at 25 o C (room
temperature) for 15 minutes.
5. The optical density of each well is then measured at 540 nm.
Rev 8/15/2008 Page 3 of 6

OD
GLYCEROL STANDARD CURVE
Generate standard curve: see example below
[DO NOT use this standard curve to generate your data. This is an example.]
Subtract the OD value of the 0 M standard from all OD values including the standard curve.
Zero
(blank) = .040
M
Glycerol OD
OD -
blank
3.125 0.054 0.014
6.25 0.066 0.026
12.5 0.082 0.042
25 0.138 0.098
50 0.214 0.174
100 0.402 0.362
200 0.711 0.671
slope = 0.0034
intercept= 0.0075
r 2 = 0.9985
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Standard Curve
y = 0.0034x + 0.0075
R 2 = 0.9985
0 50 100 150 200 250
Glycerol in uM
y = observed O.D. minus the blank
x = concentration of glycerol in M
To calculate x for each y, (i.e. to change the observed O.D. into glycerol concentration) use the following
equation:
y=(slope) times (x) plus intercept
y=mx+b so x=(y-b)/m
x=(y – 0.0075)/0.003 where 0.003= slope of the line and 0.0075= y intercept. Be careful to enter the proper sign
for the y intercept value as it may be a negative number.
Any OD values greater than the highest standard (200 M) should be suspect. The compound should be reassayed
using a lower dose of the compound at treatment OR a dilute solution of the condition medium at the
time of the assay.
The R 2 value should be equal or greater than 0.98 for the standard curve to be valid. Any R 2 values below
0.98, must have the standard curve run again.
Data are expressed as M glycerol.
REMEMBER TO ACCOUNT FOR THE DILUTION FACTOR IN STEP 3.
Rev 8/15/2008 Page 4 of 6

A
B
C
D
E
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
APPENDIX A: PLATE LAYOUT
Rev 8/15/2008 Page 5 of 6

APPENDIX B: PROCEDURE FLOWCHART
Glycerol Detection
Add 20 l/well test sample and 30 l/well dilution
buffer to one of the blank assay plates provided.
Add 50 l/well diluted standard curve to empty wells.
Sample
20 l
Plate A
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
Reconstitute Glycerol Reagent A.
Add 50 l/well.
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
OOOOOOOOOOOO
50 l/well
Glycerol
Reagent A
OOO
OOO
OOO
OOO
OOO
Plate B
Incubate 15 minutes @ room temperature.
Measure the optical density of each well
at 540 nm using a spectrophotometer
plate reader.
Reminder: Sample was diluted in Step 3
Rev 8/15/2008 Page 6 of 6